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Yang et al. (2016). “Dairy manure for bioethanol,” BioResources 11(1), 1240-1254 1240
Dairy Manure as a Potential Feedstock for Cost-Effective Cellulosic Bioethanol
Qiang Yang, Shengfei Zhou, and Troy Runge *
This study investigated sulfite pretreatment to overcome recalcitrance of lignocelluloses (SPORL) pretreatment and subsequent enzymatic digestibility of undigested dairy manure to preliminarily assess its potential use as an inexpensive feedstock for cellulosic bioethanol production. The sulfite pretreatment was carried out in a factorial analysis using 163 to 197 °C for 3 to 37 min with 0.8% to 4.2% sulfuric acid combined with 2.6% to 9.4% sodium sulfite. These treatments were compared with other standard pretreatments of dilute acid, and hot and cold alkali pretreatments. This comparative study showed that the sulfite pretreatment, through its combined effects of hemicellulose and lignin removal and lignin sulfonation, is more effective than the diluted acid and alkali pretreatments to improve the enzymatic digestibility of dairy manure.
Keywords: Dairy manure; Sulfite pretreatment; Bioethanol; Enzymatic hydrolysis
Contact information: Department of Biological Systems Engineering, University of Wisconsin-Madison,
460 Henry Mall, Madison, WI 53706, USA; *Corresponding author: [email protected]
INTRODUCTION
Cost-effective cellulosic ethanol can be produced from non-food and low-cost
agricultural residues, such as wood residuals, corn stover, and perennial grasses (Zhang et
al. 2013; Papa et al. 2015). Manure is an inexpensive lignocellulosic biomass because it
is an animal feedlot waste and is concentrated at the farm, largely eliminating the need
for transportation (Chen et al. 2005; Teater et al. 2011). More than 110 million tons of
dairy manure is produced annually in the United States (Yue et al. 2010). Traditionally,
manure is spread on agricultural fields as fertilizer. However if over-applied, it can cause
environmental issues such as watershed pollution, high nitrogen and phosphorous soil
loads, and generation of greenhouse gases (Gollehon et al. 2000).
Growing environmental concerns coupled with higher energy prices have recently
led to a renewed interest in the utilization of manure to produce economically feasible
bioenergy. Manure can be directly burned to produce heat, converted to bio-oil, or made
into combustible syngas (Fernandez-Lopez et al. 2015). Alternatively, manure can be
anaerobically digested to methane, which has a similar heating value to ethanol (Nasir et
al. 2012). However, the remaining cellulose in manure is usually underutilized by
anaerobic digestion. In fact, the anaerobically digested manure has more favorable
compositional properties (less hemicellulose and similar or more cellulose) than other
lignocellulosic biomasses, such as switchgrass (Yue et al. 2010). Technically, cellulosic
bioethanol can be biologically produced from manure through fermentation of glucose
derived from enzymatic hydrolysis of cellulose. Several studies have recently
demonstrated the potential of manure as feedstock for cost-effective cellulosic bioethanol
production (Yue et al. 2010; MacLellan et al. 2013; Zhang et al. 2013; Elumalai et al.
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Yang et al. (2016). “Dairy manure for bioethanol,” BioResources 11(1), 1240-1254 1241
2014; Vancov et al. 2015). Like other lignocelluloses, an effective pretreatment is
necessary for manure to remove recalcitrance to enzymatic hydrolysis of cellulose caused
by hemicellulose and lignin. To this end, acid and alkali pretreatments have been applied
to manure at elevated temperatures (Wen et al. 2004; Liao et al. 2006; Yue et al. 2010;
MacLellan et al. 2013; Zhang et al. 2013; Elumalai et al. 2014; Vancov et al. 2015).
However, several comparative studies have shown that sulfite pretreatment, usually
referred to as the sulfite pretreatment to overcome recalcitrance of lignocelluloses
(SPORL), is generally more effective than acid and alkaline pretreatments to improve
enzymatic digestibility of lignocellulosic biomass, such as switchgrass and spruce (Shuai
et al. 2010; Li et al. 2012; Yang and Pan 2012; Zhang et al. 2013). The sulfite
pretreatment also can offer other advantages, such as better carbohydrate recovery, less
fermentation inhibitors, and great scalability over dilute acid and alkali pretreatments
(Zhu et al. 2009).
To the best of our knowledge, there is no report on the sulfite pretreatment of
manure for cellulosic bioethanol production. Therefore, this study focuses on the sulfite
pretreatment of undigested dairy manure. Specifically, the objectives of this study were
as follows: 1) to investigate the effects of pretreatment conditions (temperature, time,
sulfuric acid, and sodium sulfite loadings) on the yield of cellulose and removal of lignin
and hemicelluloses; 2) to compare dilute acid and alkali pretreatments; 3) to evaluate the
enzymatic digestibility of pretreated dairy manure; and 4) to investigate the effects of
pretreatment conditions on the enzymatic digestibility.
EXPERIMENTAL
Materials Undigested dairy manure samples were collected from the Maple Leaf Dairy
(Cleveland, WI), and were air-dried and ground to pass a 40-mesh screen using a Wiley
mill. Sodium sulfite, sulfuric acid, urea, thiourea, and sodium hydroxide (50% w/w) were
purchased from Thermo Fisher Inc. (Waltham, MA). Polyethylenimine (PEI),
polyethylene glycol (PEG), tetracycline chloride, sodium acetate trihydrate, acetic acid,
formic acid, levunilic acid, hydroxymethylfurfural (HMF), and furfural were purchased
from Sigma-Aldrich (St. Louis, MO). Cellulase complex (Cellic CTec2) with activity of
120 filter paper units (FPU)/g was generously supplied by Novozymes (Franklinton, NC).
Pretreatments Sulfite, dilute acid, and hot alkali pretreatments were conducted at temperatures
ranging from 163 to 197 °C in 100-mL plastic vessels in batch modes using a microwave
reactor (Mars, CEM Corp., Matthews, NC). Pretreatment liquors were prepared by
mixing the required chemicals (sulfuric acid, sodium sulfite, and sodium hydroxide) with
water. Chemical loading was based on the oven dry weight of raw manure. The
pretreatment liquors containing 0.8% to 4.2% sulfuric acid and 2.6% to 9.4% sodium
sulfite were used for the sulfite pretreatment, and the pretreatment liquors with 4.0%
sulfuric acid and 8.0% sodium hydroxide were used for the dilute acid and hot alkali
pretreatments, respectively. Briefly, 10 g of manure (oven dried) was mixed with 60 mL
of pretreatment liquor. Pretreated manure was collected through filtration and thoroughly
washed with deionized water. All pretreated manure samples were stored at 4 °C before
enzymatic hydrolysis.
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The sulfite pretreatment experiment was specially designed using Design-Expert
(DX7, 2005) software with response surface methodology (RSM) combined with Hartley
composite design (eight factorial points, eight star points, and five center points with a
specified alpha value of 1.7 as an axial scaling). The obtained experimental data were
further analyzed by JMP Pro 11 software (SAS Institute, Cary, NC) to establish fitted
models, and then the three-dimensional surface plots were generated using MATLAB
R2014 software (Math Works, Natick, MA), based on established models. The sulfite
pretreatment conditions were chosen according to those reported for switchgrass and
woody biomass in the literature with slight modifications to accommodate the manure
biomass (Shuai et al. 2010; Yang and Pan 2012; Zhang et al. 2013). Specifically, the
pretreatment experiments were composed of 21 runs, and the pretreatments were carried
out at temperature ranges of 163 to 197 °C for 3 to 37 min using 0.8% to 4.2% sulfuric
acid combined with 2.6% to 9.4% sodium sulfite, as summarized in Table 1. The alkali
pretreatments at a cold temperature were carried out in 250-mL flasks at -20 °C. The
alkali aqueous solutions were prepared by mixing 7.0 wt.% sodium hydroxide with
12.0% urea, 5.5% thiourea, 2.0% polyethylenimine, or 2.0% polyethylene glycol
(Mohsenzadeh et al. 2012). Briefly, 10 g of manure (oven-dried) was mixed with 50 mL
of alkali aqueous solution, and the resultant mixture was stirred for 30 min and then
stored at – 20 °C for 12 h. Then, 100 mL of deionized water was added and stirred for 1
h. Pretreated manure was recovered through filtration and thoroughly washed with
deionized water.
Enzymatic Hydrolysis Enzymatic hydrolysis was carried out in a 50-mL conical tube at 50 °C on a
shaking incubator (New Brunswick Scientific Excella E24, Edison, NJ) at 180 rpm using
40 mL of sodium acetate buffer (50 mM, pH 4.8) at 2.0% consistency containing 2.0 mg
of tetracycline chloride to deter bacterial action. The cellulase complex loading was 20
FPU/g of cellulose. Aliquots (0.4 mL) were taken periodically for glucose analysis, and
sodium acetate buffers (0.4 mL, 50 mM, pH 4.8) were added to make up the volume.
Analytic Methods Ash, extractives, and acid-insoluble (Klason) lignin were analyzed according to
National Renewable Energy Laboratory (NREL) analytic procedures (Sluiter et al. 2010).
Acid-soluble lignin was measured using a UV-vis spectrophotometer (Cary 50 Bio,
Varian, Santa Clara, CA) at 205 nm with an extinction coefficient of 110 L g-1cm-1.
Monosaccharides (glucose, galactose, arabinose, mannose, and xylose) were measured
using high-performance ion chromatography (HPIC, ICS-3000, Dionex, Sunnyvale, CA,
USA) equipped with an integrated amperometric detector and Carbopac guard (PA20, 3 ×
30 mm) and analytic (PA20, 3 × 150 mm) columns. Fermentation inhibitors (acetic acid,
formic acid, levunilic acid, hydroxymethylfurfural, and furfural) were measured using the
Dionex ICS-3000 system (Sunnyvale, CA), equipped with a UV-vis detector and
Superlcogel C-610H analytic (30 cm × 7.8 mm) and guard (5 cm × 4.6 mm) columns.
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RESULTS AND DISCUSSION Sulfite Pretreatment
The sulfite pretreatment was evaluated in terms of hemicellulose and lignin
removal, concomitant changes in biomass components, substrate yield, and fermentation
inhibitors produced. Cellulose and hemicellulose are expressed in terms of their
monosaccharides rather than polysaccharides in this study, as saccharification was the
final product. Component analysis showed that the dairy manure contained 23%
cellulose, 23.5% lignin, 18% xylose, as well minor saccharides of 1.7% galactose, 2.3%
arabinose, and 0.1% mannose.
The undigested dairy manure in this study had slightly more hemicellulose than
reported digested manures, which was attributed primarily to the cows’ diets (Yue et al.
2010; Teater et al. 2011; Vancov et al. 2015). Ash and extractives were 14.6% and
16.6%, respectively. It is well-established that hemicellulose and lignin can be removed
through acid-catalyzed depolymerization reactions.
Effects of the pretreatment conditions on lignin and hemicellulose removal were
determined and shown in Fig.1. As summarized in Table 1, the sulfite pretreatment under
different conditions removed 11.6% to 38.3% lignin and 18.1% to 67.7% hemicellulose.
Pretreatment temperature and time were set at their median values, when investigating
effects of sulfuric acid and sodium sulfite loadings, and vice-versa.
Fig. 1. Effects of pretreatment conditions on lignin and hemicellulose removals during sulfite pretreatment of manure
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Table 1. Sulfite Pretreatment of Manure
Note: SS: sodium sulfite; SA: sulfuric acid; Gla: galactose; Ara: arabinose; Xyl: xylose; Glu: glucose; KL: Klason lignin; CGC: cellulose-to-glucose conversion; SL: soluble lignin; AC: acetic acid. Chemicals (SS and SA), % on oven-dry manure; apH of sulfuric acid aqueous solution; bpH of sulfuric acid aqueous solution with sodium sulfite; ccomposition (xylose, glucose or lignin), % on dry pretreated manure; dcomposition (glucose, soluble lignin, xylose or acetic acid), % on dry untreated manure
Exp. No. Pretreatment conditionsa Yield,
%
Pretreated substratec CGC at 72 h,
%
Pretreatment spent liquord
Temp, °C
Time, min
SA, %
pHa SS, %
pHb Gla, %
Ara, %
Xyl, %
Glu, %
KL, %
Glu, %
SL, %
Xyl, %
AC, %
pH
1 180 20 2.5 1.86 6.0 5.43 56.7 2.4 2.4 20.2 36.5 23.4 57.7 0.2 6.6 6.2 1.0 6.44
2 197 20 2.5 1.86 6.0 5.43 48.8 1.9 1.4 11.5 36.7 26.4 62.3 0.5 5.3 12.1 1.1 6.52
3 163 20 2.5 1.86 6.0 5.43 69.6 3.1 2.9 20.7 31.6 24.3 36.6 0.1 5.9 3.9 0.6 6.88
4 190 10 3.5 1.78 8.0 2.93 8.2 2.1 1.8 20.9 38.8 26.4 56.8 0.1 6.3 4.9 0.9 6.61
5 170 10 3.5 1.78 4.0 2.09 72.0 2.1 2.4 20.1 33.4 24.6 36.8 0 5.5 3.3 0.8 6.91
6 180 37 2.5 1.86 6.0 5.43 45.4 1.9 2.1 19.2 35.1 24.7 57.6 0.7 6.0 8.9 1.0 6.65
7 170 30 1.5 1.91 8.0 6.63 65.0 2.3 2.1 19.5 36.5 22.6 34.9 0 5.9 6.8 0.8 6.23
8 180 20 2.5 1.86 6.0 5.43 57.9 1.7 1.6 15.9 39.1 25.8 52.1 0.1 5.7 4.1 1.1 6.66
9 180 20 2.5 1.86 6.0 5.43 56.2 2.5 2.2 19.3 35.8 24.6 38.5 0.3 4.7 9.2 0.8 6.92
10 180 3 2.5 1.86 6.0 5.43 71.3 2.1 1.9 19.1 32.0 25.6 46.8 0.1 5.2 9.6 0.8 6.78
11 180 20 2.5 1.86 6.0 5.43 55.7 1.5 1.5 14.9 38.9 25.4 48.5 0.1 6.3 7.1 0.9 6.88
12 190 30 1.5 1.91 4.0 6.69 55.4 1.4 1.4 14.6 37.4 28.3 55.4 0.2 4.4 10.1 3.6 6.44
13 180 20 2.5 1.86 2.6 2.12 61.9 2.0 2.0 17.0 30.5 27.7 34.6 0.4 4.2 6.1 0.7 6.43
14 180 20 2.5 1.86 9.4 6.26 42.3 2.2 2.0 18.3 36.1 22.3 48.3 0.8 6.4 9.2 0.7 6.60
15 170 30 3.5 1.78 8.0 2.83 62.2 2.1 1.8 18.4 35.5 25.1 36.3 0.1 2.7 6.0 0.6 6.58
16 180 20 4.2 1.71 6.0 2.15 52.6 1.8 1.9 16.3 33.7 28.1 48.8 0.5 5.1 9.2 0.7 6.71
17 180 20 2.5 1.86 6.0 5.43 56.4 2.3 1.9 21.2 37.7 26.5 42.2 0.2 7.5 6.3 0.7 6.98
18 180 20 0.8 1.99 6.0 6.62 61.0 2.0 1.7 18.3 36.9 23.9 51.4 0.1 7.2 6.5 6.0 6.57
19 190 10 1.5 1.91 8.0 6.63 63.4 1.3 1.2 15.9 35.7 23.7 54.9 0.1 9.0 7.6 5.9 6.69
20 190 30 3.5 1.78 4.0 2.09 52.5 0.7 0.7 12.2 38.5 34.6 49.6 0.3 4.5 10.3 0.9 6.34
21 170 10 1.5 1.91 4.0 6.69 77.2 2.0 1.8 17.2 30.1 26.7 38.5 0 5.6 4.3 0.4 6.60
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As shown in Fig. 1, the hemicellulose and lignin removals were directly affected
by the pretreatment conditions (temperature, time, sulfuric acid, and sodium sulfite
loadings). The results showed that more lignin and hemicellulose can be removed under
higher temperatures for longer time periods. In this study, both the pretreatment
temperature and time were critical for hemicellulose removal. For instance, hemicellulose
can be removed most when pretreated for 40 min at 200 °C. Lignin removal was more
dependent upon the pretreatment time, because the least lignin removal can be obtained
when the pretreatment is carried out at 195 °C for only 4 min. Also, more hemicellulose
can be removed by increasing the dosage of sulfuric acid through accelerating acid-
catalyzed hydrolysis of hemicellulose. However, sodium sulfite notably decreased the
acidity of dilute acid aqueous solution, as evidenced by the fact that pH value greatly
increased after addition of sodium sulfite. Therefore, the presence of sodium sulfite was
not favorable to the acid-catalyzed hydrolysis of hemicellulose (Yang and Pan 2012;
Zhang et al. 2013). Unexpectedly, it seems that more hemicellulose in this study can be
removed with more sodium sulfite; however, minimal hemicellulose removal (18%) was
observed even when the pretreated of sodium sulfite (with 2.5% sulfuric acid) was 6.0%
at 163 °C for 20 min. This result indicates that the pretreatment temperature and time are
more critical for the hemicellulose removal in this study. The acid-catalyzed lignin
removal was accomplished mostly through the cleavage of α- and β-ether linkages (Cao
et al. 2012). Therefore, the addition of more sulfuric acid resulted in more lignin removal.
However, lignin depolymerization was accompanied through lignin condensation during
the formation of intra-linkages, such as carbon-carbon bonding (Cao et al. 2012).
Furthermore, the lignin condensation reaction tended to be more pronounced with
increasing dosages of sulfuric acid. On one hand, sodium sulfite can facilitate lignin
removal through sulfonation; increasing the dosage of sodium sulfite was generally
beneficial for lignin removal in this study. On the other hand, a surplus sodium sulfite can
hinder lignin removal by decreasing the acidity of the sulfuric acid aqueous solution. For
example, when the dosage of sulfuric acid was in the range of 4.0% to 6.0%, further
increasing the dosage of sodium sulfite from approximately 8.0% to 10.0% resulted in a
decrease in lignin removal.
Removed lignin and hemicellulose found in the spent liquors respectively existed
in the forms of soluble lignin and monosaccharides (xylose and glucose). Under severe
acidic conditions at elevated temperature, xylose and glucose can be thermally degraded
into furfural and HMF, respectively (Oefner et al. 1992; Woo et al. 2015). The acidity of
the pretreatment liquor is decided by the dosages of sulfuric acid and sodium sulfite. As
shown in Table 1, some pretreatment liquors with pH values of 2 were very acidic, while
others are weakly acidic. However, when buffered by sodium sulfite and formed acetic
acid by acidic hydrolysis of acetyl groups in hemicellulose, the pretreatment spent liquors
exhibited similarity in pH value of 6.6 and were weakly acidic. Therefore, only small
amounts of furfural (about 4.0%) and HMF (about 0.3%) were found in the pretreatment
spent liquors. Although furfural may inhibit fermentation, the SPORL pretreatment may
be low enough to not cause as much inhibition as dilute acid or alkali pretreatments.
Because of the removal of lignin and hemicellulose, the pretreated manures were
enriched with cellulose (30.5 to 41.7%) relative to untreated manure (23%). Nevertheless,
the pretreated manures still exhibited higher amounts of lignin (22.3% to 34.6%),
primarily because of the removal of hemicellulose. In this study, the hemicellulose and
lignin removals caused the manure substantial losses in mass during the sulfite
pretreatment. The manure yield ranged from 42.3% to 77.2%. As discussed above,
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increasing the treatment temperature was favorable for the removal of lignin; however,
the condensation of lignin can be likely pronounced as the pretreatment prolongs.
Therefore, increasing temperature and extending pretreatment time can enhance
dissolution of hemicellulose and even cellulose. Consequently, higher substrate yields
can be achieved under either lower temperature or shorter treatment duration. For
example, when the dosages of sodium sulfite and sulfuric acid were 6.0% and 2.5%,
respectively, higher substrate yields (48.8% to 71.3%) were achieved under either shorter
time (e.g., 71.3% for run 10) or lower temperatures (e.g., 69.6% for run 3). Increasing the
loading of sulfuric acid or/and sodium sulfite can result in a decreased substrate yield
because of the accelerated hemicellulose hydrolysis or/and delignification. Therefore,
higher substrate yields are achieved under less sulfuric acid or/and sodium sulfite. For
example, lower substrate yields (42.3% to 45.4%) were observed when the pretreatments
(runs 6 and 14) were carried out at 180 °C for 20 to 37 min, using higher (2.5%) sulfuric
acid addition, combined with greater (6.0% to 9.4%) sodium sulfite inclusion. The
highest substrate yield (77.2%) in this study was achieved under mild pretreatment
conditions (170 °C, 10 min, 4.0% sodium sulfite, and 1.5% sulfuric acid).
Enzymatic Hydrolysis The sulfite-pretreated manures were then compared in enzymatic digestibility
trials using untreated manure as a control. The time-dependent hydrolysis profiles,
presented in Fig. 2, indicate that all pretreated manures showed higher initial hydrolysis
rates (conversion at first hour) than untreated manure, and they exhibited much better
performance as the hydrolysis progressed. To allow for facile comparison, cellulose-to-
glucose conversions (CGC), after 72 h of hydrolysis, are summarized in Table 1. The
data indicate that the untreated manure had a very poor enzymatic digestibility, with only
approximately 3.0% of the cellulose enzymatically converted into glucose after 72 h of
hydrolysis. By comparison, the sulfite-pretreated manures were much more digestible,
with 36.6% to 62.3% CGCs after the same enzyme loadings. These results show that the
sulfite pretreatment can noticeably improve the enzymatic digestibility of manure. The
removal of lignin and hemicellulose was hypothesized to be responsible for the
improvement in enzymatic digestibility by creating accessibility for enzymes to
hydrolyze cellulose (Leu and Zhu 2013; Zhang et al. 2013; Meng and Ragauskas 2014).
0 10 20 30 40 50 60 70
0
10
20
30
40
50
60
70
Ce
llulo
se-t
o-g
luco
se c
onve
rsio
n (
%)
Hydrolysis time (h)
1 2 3 4 5 6 7 8
9 10 Untreated
0 10 20 30 40 50 60 70
0
10
20
30
40
50
60
70
Cellu
lose-t
o-g
lucose c
onvers
ion (
%)
Hydrolysis time (h)
11 12 13 14 15 16 17
18 19 20 21 Untreated
Fig. 2. Time-dependent enzymatic hydrolysis profiles of sulfite-pretreated manure experiments described in Table 1 (legends refer to experiment number).
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Fig. 3. Effects of lignin or hemicellulose removal on cellulose-to-glucose conversion (CGC) after 72 h in sulfite-pretreated manure
Fig. 4. Effects of pretreatment conditions on cellulose-to-glucose conversion (CGC) after 72 h in sulfite-pretreated manure
As shown in Fig. 3, the enzymatic digestibility (CGC at 72 h) of manure in this
study was positively correlated with the removal of lignin and hemicellulose. The effects
of the pretreatment conditions on the subsequent enzymatic digestibility (CGC at 72 h)
are shown in Fig. 4. Increasing the pretreatment temperature or/and prolonging the
pretreatment were beneficial to the enzymatic hydrolysis because of the enhanced
removal of lignin and hemicellulose. For example, the manures pretreated at 180 to
197 °C generally achieved higher cellulose-to-glucose conversions (> 45%) than those
pretreated at 163 to 170 °C (< 39%). Moreover, it seemed that increasing the
pretreatment temperature was more effective than prolonging pretreatment. For example,
the manure pretreated at 180 °C for only 3 min achieved a 46.8% CGC after 72 h of
hydrolysis. This was likely because a higher temperature is necessary to initiate the acid-
catalyzed delignification. The effect of sodium sulfite or sulfuric acid loading on the
enzymatic digestibility is complicated, as shown in Fig. 4. In general, increasing the
loading of sulfuric acid in the presence of sodium sulfite can improve the enzymatic
digestibility by removing more hemicellulose and lignin. Similarly, increasing the dosage
of sodium sulfite at a constant sulfuric acid loading rate can also improve the digestibility
by enhancing the lignin removal through sulfonation. However, this was not always the
case, as surplus sodium sulfite can also reduce the hemicellulose removal by decreasing
the acidity of the pretreatment liquor. As discussed earlier, the hemicellulose removal in
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this study was more critical than the lignin removal, as shown in Fig. 3. Therefore, there
should be a trade-off between the acid-catalyzed hemicellulose removal and sulfonation-
caused lignin removal by increasing the loading of sodium sulfite to improve the
enzymatic digestibility in this study.
Comparison of Sulfite, Diluted Acid, and Alkali Pretreatments
Diluted acid and alkali pretreatments are among the most common investigated
routes for enhancement of cellulosic bioethanol production (Agbor et al. 2011). The
dilute acid and alkali pretreatments were therefore investigated and compared with the
sulfite pretreatment in this study. It has been well-recognized that sodium hydroxide, at
high temperatures, can remove lignin through the cleavage of linkages, such as β-O-4.
Sodium hydroxide hydrates at a high concentration can also penetrate the amorphous
regions of cellulose, further altering the neighboring crystalline regions; it can even
hydrolyze cellulose through a peeling reaction from the reducing end (Cai and Zhang
2005). At high concentrations and cold temperatures, some chemicals, such as urea,
thiourea, polyethylene glycol (PEG), and polyethylenimine (PEI), can facilitate the
dissolution of cellulose by interrupting or destroying the cellulose hydrogen bonds (Zhao
et al. 2008; Mohsenzadeh et al. 2012). Therefore, in this study, sodium hydroxide alone
was used for the hot alkali pretreatment, and sodium hydroxide combined with urea,
thiourea, PEG, or PEI was used for the cold alkali pretreatment. The conditions for hot
and cold alkali pretreatments were chosen according to the literature with modifications
(Mohsenzadeh et al. 2012; Yang and Pan 2012; Zhang et al. 2013). Eight percent sodium
hydroxide (based on oven-dry manure) was used for the hot alkali pretreatment, while
7.0% sodium hydroxide aqueous solutions containing one of the following: 12% urea,
5.5% thiourea, 2.0% PEG, or 2.0% PEI were used for the cold alkali pretreatments. The
hot alkali pretreatment was carried out at 180 °C for 30 min, and the cold alkali
pretreatments were conducted at -20 °C for 12 h. For comparison, the diluted acid
pretreatment was also carried out at 180 °C for 30 min, and 4.0% sulfuric acid was used.
Table 2. Pretreatment Conditions of Manure using Sulfite, Diluted Acid, and Alkali
Pretreatment Temp (°C)
Time (h)
Chemicals
Sulfitea 180 0.5 9.0% sodium sulfite + 4.0% sulfuric acid
Diluted acida 180 0.5 4.0% sulfuric acid
Alkali-1a 180 0.5 8.0% sodium hydroxide
Alkali-2 -20 12 7.0% sodium hydroxide + 2.0% polyethylenimine
Alkali-3 -20 12 7.0% sodium hydroxide + 2.0% polyethylene glycol
Alkali-4 -20 12 7.0% sodium hydroxide + 5.5% thiourea
Alkali-5 -20 12 7.0% sodium hydroxide + 12% urea aChemical loading was based on the oven-dry weight of manure
As shown in Fig. 4, sodium sulfite improved the digestibility of sulfite-pretreated
manure. Therefore, 9.0% sodium sulfite was chosen, and to compare with the diluted
acid, the same amount of sulfuric acid (4.0%) was also used for the sulfite pretreatment,
which was also conducted at 180 °C for 30 min. The presence of 9.0% sodium sulfite
increased the pH value of the pretreatment liquor loaded with 4.0% sulfuric acid from
1.75 to 2.52. The sulfite, dilute acid, and alkali pretreatment conditions are summarized
in Table 2, and the pretreatment results are shown in Fig. 5.
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Fig. 5. Comparison of sulfite, dilute acid, and alkali pretreatments of manure
As expected, the dilute acid pretreatment removed more (74%) hemicellulose than
the sulfite (45.7%), hot alkali (42.9%), or cold alkali (about 40%) pretreatments. As a
result, the dilute acid-pretreated manure exhibited less hemicellulose (10.4%) than the
sulfite (20.1%), hot alkali- (24.3%), or cold alkali- (21.9 to 22.3%) pretreated manures.
The sulfite pretreatment spent liquor (pH=6.89) contained 5.0% and 0.4% furfural and
HMF, respectively. Because of its stronger acidity (pH=4.92), more furfural and HMF
(8.0% and 0.6%, respectively) were observed in the dilute acid pretreatment spent liquor.
Therefore, in the pretreatment spent liquors, more xylose and glucose were found for the
cold alkali (14.8% to 15.2%) and the hot alkali (11.1%) pretreatments than the dilute acid
(4.3%) and the sulfite (3.8%) pretreatments. The hot alkali pretreatment removed more
(63.8%) lignin than the cold alkali pretreatment (54% to 58%). Accordingly, more
soluble lignin was observed in the hot alkali pretreatment spent liquor.
During the sulfite pretreatment, lignin can be removed through the acid-catalyzed
cleavage of lignin linkages, as well as through sulfonation. Therefore, the sulfite
pretreatment removed more (54% vs. 45%) lignin than the dilute acid pretreatment. The
sulfite and cold alkali pretreated manures exhibited similar levels (about 22%) of lignin.
However, the hot alkali-pretreated manure contained the least lignin (20%), while the
dilute acid-pretreated manure had the highest lignin level (28.7%), which was attributed
to greater removal of hemicellulose. Apparently, the removal of hemicellulose and lignin
both influenced the substrate yield. These results showed that the sulfite (48.6%), dilute
acid (45.2%), and cold alkali (45.4% to 48.3%) pretreatments resulted in relatively higher
substrate yields than the hot alkali pretreatment (42.3%).
After lignin and hemicellulose are partially removed, manure is enriched in
cellulose. The hot alkali pretreatment resulted in higher glucose conversion (40.1%) than
the cold alkali one (about 31%), likely because more lignin was removed. Cellulose can
be also hydrolyzed by sulfuric acid; however, sodium sulfite can alleviate the acid-
catalyzed hydrolysis through decreasing the acidity (Yang and Pan 2012; Zhang et al.
2013). As a result, at the same sulfuric acid loading level, the sulfite-pretreated manure
had more (37.5%) cellulose than the diluted acid-pretreated manure (34.9%). Acetic acid
was observed in the diluted acid and sulfite pretreatment spent liquors, but it did not
appear in the alkali pretreatment spent liquors, likely because it was neutralized by
sodium hydroxide. The organic acids neutralizing the pH were observed with the pH
value of hot alkali pretreatment liquor, which dropped from 11.7 to 9.1 (in the spent
liquor) after pretreatment.
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Yang et al. (2016). “Dairy manure for bioethanol,” BioResources 11(1), 1240-1254 1250
The results for enzymatic digestibility of manures pretreated by sulfite, diluted
acid, and alkali are comparatively shown in Figs. 5 and 6. It was apparent that the sulfite-
pretreated manure was hydrolyzed at the fastest rate and achieved the best enzymatic
digestibility. A 53.5% cellulose-to-glucose conversion was obtained after 72 h of
hydrolysis, which was close to the predicted value (54.4%) based on the fitted model (a
function of temperature, time, sodium sulfite, and sulfuric acid) generated by JMP.
Moreover, it can be concluded from Fig. 4 that the performance of sulfite pretreatment
could be further improved under higher temperature for a longer time. For example, a
71% cellulose-to-glucose conversion after 72 h of hydrolysis was achieved when the
pretreatment was carried out at 190 °C for 35 min by 9.0% sodium sulfite and 4.0%
sulfuric acid. In contrast, the diluted acid-pretreated manure was less hydrolyzed, and
therefore achieved a lower cellulose-to-glucose conversion (45%). However, the sulfite-
pretreated manure had almost twice (20.1%) the hemicellulose than the dilute acid-
pretreated manure (10.4%). The better digestibility for the sulfite-pretreated manure can
be primarily attributed to its lower lignin content (22.1% vs. 28.7%). Looking at other
biomass SPORL treatments, the residual lignin in the sulfite pretreatment was found to be
less condensed than that of the diluted acid pretreatment, which should make the lignin
more hydrophilic for the sulfite treatment (Rahikainen et al. 2013). Because of the
weaker acidity and sulfonation, it can be reasonably hypothesized that the residual lignin
in the sulfite-pretreated manure was less condensed and less hydrophobic than that of the
dilute acid-pretreated manure.
Fig. 6. Time-dependent enzymatic hydrolysis profiles of manure pretreated by sulfite, diluted acid, and alkali
Lignin adsorbs cellulolytic enzymes primarily through hydrophobic and ionic
interactions (Eriksson et al. 2002). Therefore, with less content of lignin and less
hydrophobic lignin, sulfite-pretreated lignocelluloses, such as switchgrass and agave
substrates, can adsorb less cellulolytic enzymes compared with dilute acid-pretreated
ones (Yang and Pan 2012; Zhang et al. 2013). The residual lignin with reduced
hydrophobicity can adsorb less cellulolytic enzymes, leaving more enzymes for digesting
cellulose. Therefore, the partial sulfonation of residual lignin might also contribute to the
better digestibility of the sulfite-pretreated manure in this study. In comparison, the hot
alkali-pretreated manure showed the poorest digestibility, with an almost 30.3% cellulose
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Yang et al. (2016). “Dairy manure for bioethanol,” BioResources 11(1), 1240-1254 1251
conversion after 72 h of hydrolysis, even though it had the lowest content (20%) of
lignin. This result could have been caused by its higher content (24.3%) of hemicellulose.
The hot alkali pretreatment also resulted in a poor enzymatic digestibility for switchgrass
(Zhang et al. 2013). Conversely, the cold alkali-pretreated manures, with relatively
higher contents of lignin (about 22%) and similar contents (about 24%) of
hemicelluloses, exhibited favorable enzymatic digestibility, with over 40% cellulose
conversion.
It has been reported that cold alkali pretreatment (sodium hydroxide combined
with urea, thiourea, or PEG) may reduce the crystallinity of cellulose in both spruce and
birch woods (Zhao et al. 2008). Glycosidic bonds in crystalline cellulose are protected by
their strong inter- and intra-molecular hydrogen bonding. However, the disruption of
hydrogen bonds (reduction in the crystallinity) can expose more β-1,4 glycosidic bonds
for enzymatic hydrolysis. Therefore, it can be assumed that cellulose in the cold alkali-
pretreated manure had a reduced crystallinity, and consequently showed favorable
digestibility.
CONCLUSIONS
1. Sulfite pretreatment outperformed dilute acid (with same amount of sulfuric acid) and
hot and cold (more chemicals and longer time) alkali pretreatments.
2. Sulfite pretreatment also exhibited other advantages, such as better sugar recovery
and less fermentation inhibitors over the dilute acid pretreatment, and less chemicals
required and shorter reaction times over the cold alkali pretreatment.
3. Sulfite pretreatment can notably improve the enzymatic digestibility of undigested
dairy manure by reducing the recalcitrance through the removal of hemicellulose and
lignin in the lignocellulosic matrix.
4. This study suggests that undigested dairy manure can be considered as a potential
feedstock for cost-effective cellulosic bioethanol production. Further development
and economic analysis will likely be required prior to commercialization.
ACKNOWLEDGMENTS
The authors are grateful for the support of the United States Department of
Agriculture-National Institute of Food and Agriculture (USDA BRDI Grant number:
2012-10006-19423) and the Wisconsin Energy Institute for their financial support. We
thank Dr. Xuejun Pan for his assistance with the microwave reactor for pretreatment
experiments.
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Article submitted: September 2, 2015; Peer review completed: November 22, 2015;
Revisions received and accepted: November 28, 2015; Published: December 14, 2015.
DOI: 10.15376/biores.11.1.1240-1254